Clonal structure and the specificity of vaccine-induced T cell response to SARS-CoV-2 Spike protein.

Adenovirus vaccines, particularly the COVID-19 Ad5-nCoV adenovirus vaccine, have emerged as promising tools in the fight against infectious diseases. In this study, we investigated the structure of the T cell response to the Spike protein of the SARS-CoV-2 virus used in the COVID-19 Ad5-nCoV adenoviral vaccine in a phase 3 clinical trial (NCT04540419). In 69 participants, we collected peripheral blood samples at four time points after vaccination or placebo injection. Sequencing of T cell receptor repertoires from Spike-stimulated T cell cultures at day 14 from 17 vaccinated revealed a more diverse CD4+ T cell repertoire compared to CD8+. Nevertheless, CD8+ clonotypes accounted for more than half of the Spike-specific repertoire. Our longitudinal analysis showed a peak T cell response at day 14, followed by a decline until month 6. Remarkably, multiple T cell clonotypes persisted for at least 6 months after vaccination, as demonstrated by ex vivo stimulation. Examination of CDR3 regions revealed homologous sequences in both CD4+ and CD8+ clonotypes, with major CD8+ clonotypes sharing high similarity with annotated sequences specific for the NYNYLYRLF peptide, suggesting potential immunodominance. In conclusion, our study demonstrates the immunogenicity of the Ad5-nCoV adenoviral vaccine and highlights its ability to induce robust and durable T cell responses. These findings provide valuable insight into the efficacy of the vaccine against COVID-19 and provide critical information for ongoing efforts to control infectious diseases.

Breg-Mediated Immunoregulation in the Skin.

Wound healing is a complex process involving a coordinated series of events aimed at restoring tissue integrity and function. Regulatory B cells (Bregs) are a subset of B lymphocytes that play an essential role in fine-tuning immune responses and maintaining immune homeostasis. Recent studies have suggested that Bregs are important players in cutaneous immunity. This review summarizes the current understanding of the role of Bregs in skin immunity in health and pathology, such as diabetes, psoriasis, systemic sclerosis, cutaneous lupus erythematosus, cutaneous hypersensitivity, pemphigus, and dermatomyositis. We discuss the mechanisms by which Bregs maintain tissue homeostasis in the wound microenvironment through the promotion of angiogenesis, suppression of effector cells, and induction of regulatory immune cells. We also mention the potential clinical applications of Bregs in promoting wound healing, such as the use of adoptive Breg transfer.

Differentially activated B cells develop regulatory phenotype and show varying immunosuppressive features: a comparative study.

Regulatory B lymphocytes (Bregs) are B cells with well-pronounced immunosuppressive properties, allowing them to suppress the activity of effector cells. A broad repertoire of immunosuppressive mechanisms makes Bregs an attractive tool for adoptive cell therapy for diseases associated with excessive activation of immune reactions. Such therapy implies Breg extraction from the patient's peripheral blood, ex vivo activation and expansion, and further infusion into the patient. At the same time, the utility of Bregs for therapeutic approaches is limited by their small numbers and extremely low survival rate, which is typical for all primary B cell cultures. Therefore, extracting CD19+ cells from the patient's peripheral blood and specifically activating them ex vivo to make B cells acquire a suppressive phenotype seems to be far more productive. It will allow a much larger number of B cells to be obtained initially, which may significantly increase the likelihood of successful immunosuppression after adoptive Breg transfer. This comparative study focuses on finding ways to efficiently manipulate B cells in vitro to differentiate them into Bregs. We used CD40L, CpG, IL4, IL21, PMA, and ionomycin in various combinations to generate immunosuppressive phenotype in B cells and performed functional assays to test their regulatory capacity. This work shows that treatment of primary B cells using CD40L + CpG + IL21 mix was most effective in terms of induction of functionally active regulatory B lymphocytes with high immunosuppressive capacity ex vivo.

rs71327024 Associated with COVID-19 Hospitalization Reduces CXCR6 Promoter Activity in Human CD4+ T Cells via Disruption of c-Myb Binding.

Single-nucleotide polymorphism rs71327024 located in the human 3p21.31 locus has been associated with an elevated risk of hospitalization upon SARS-CoV-2 infection. The 3p21.31 locus contains several genes encoding chemokine receptors potentially relevant to severe COVID-19. In particular, CXCR6, which is prominently expressed in T lymphocytes, NK, and NKT cells, has been shown to be involved in the recruitment of immune cells to non-lymphoid organs in chronic inflammatory and respiratory diseases. In COVID-19, CXCR6 expression is reduced in lung resident memory T cells from patients with severe disease as compared to the control cohort with moderate symptoms. We demonstrate here that rs71327024 is located within an active enhancer that augments the activity of the CXCR6 promoter in human CD4+ T lymphocytes. The common rs71327024(G) variant makes a functional binding site for the c-Myb transcription factor, while the risk rs71327024(T) variant disrupts c-Myb binding and reduces the enhancer activity. Concordantly, c-Myb knockdown in PMA-treated Jurkat cells negates rs71327024's allele-specific effect on CXCR6 promoter activity. We conclude that a disrupted c-Myb binding site may decrease CXCR6 expression in T helper cells of individuals carrying the minor rs71327024(T) allele and thus may promote the progression of severe COVID-19 and other inflammatory pathologies.

Architecture of the SARS-CoV-2-specific T cell repertoire.

The T cell response plays an indispensable role in the early control and successful clearance of SARS-CoV-2 infection. However, several important questions remain about the role of cellular immunity in COVID-19, including the shape and composition of disease-specific T cell repertoires across convalescent patients and vaccinated individuals, and how pre-existing T cell responses to other pathogens-in particular, common cold coronaviruses-impact susceptibility to SARS-CoV-2 infection and the subsequent course of disease. This review focuses on how the repertoire of T cell receptors (TCR) is shaped by natural infection and vaccination over time. We also summarize current knowledge regarding cross-reactive T cell responses and their protective role, and examine the implications of TCR repertoire diversity and cross-reactivity with regard to the design of vaccines that confer broader protection against SARS-CoV-2 variants.

Structural insights into protection against a SARS-CoV-2 spike variant by T cell receptor diversity.

T cells play a crucial role in combatting SARS-CoV-2 and forming long-term memory responses to this coronavirus. The emergence of SARS-CoV-2 variants that can evade T cell immunity has raised concerns about vaccine efficacy and the risk of reinfection. Some SARS-CoV-2 T cell epitopes elicit clonally restricted CD8+ T cell responses characterized by T cell receptors (TCRs) that lack structural diversity. Mutations in such epitopes can lead to loss of recognition by most T cells specific for that epitope, facilitating viral escape. Here, we studied an HLA-A2-restricted spike protein epitope (RLQ) that elicits CD8+ T cell responses in COVID-19 convalescent patients characterized by highly diverse TCRs. We previously reported the structure of an RLQ-specific TCR (RLQ3) with greatly reduced recognition of the most common natural variant of the RLQ epitope (T1006I). Opposite to RLQ3, TCR RLQ7 recognizes T1006I with even higher functional avidity than the WT epitope. To explain the ability of RLQ7, but not RLQ3, to tolerate the T1006I mutation, we determined structures of RLQ7 bound to RLQ-HLA-A2 and T1006I-HLA-A2. These complexes show that there are multiple structural solutions to recognizing RLQ and thereby generating a clonally diverse T cell response to this epitope that assures protection against viral escape and T cell clonal loss.

Interplay between Immune Cell Infiltration and Tumor Histological Subtype: A Case of Adrenocortical Cancer.

The analysis of the tumor microenvironment, especially tumor-infiltrated immune cells, is essential for predicting tumor prognosis, clinical outcomes, and therapy strategies. Adrenocortical cancer is a rare nonimmunogenic malignancy in which the importance of the presence of immune cells is not well understood. In our study, we made the first attempt to understand the interplay between the histology of adrenocortical cancer and its immune landscape using cases from The Cancer Genome Atlas database and the Endocrinology Research Centre collection (Moscow, Russia). We showed that the oncocytic variant of adrenocortical cancer is characterized by intensive immune infiltration and better survival, and it is crucial to analyze the effect of immune infiltration independently for each histological variant.

Multi-dimensional immunoproteomics coupled with in vitro recapitulation of oncogenic NRASQ61R identifies diagnostically relevant autoantibody biomarkers in thyroid neoplasia.

Tumor-associated antigen (TAA)-specific autoantibodies have been widely implicated in cancer diagnosis. However, cancer cell lines that are typically exploited as candidate TAA sources in immunoproteomic studies may fail to accurately represent the autoantigen-ome of lower-grade neoplasms. Here, we established an integrated strategy for the identification of disease-relevant TAAs in thyroid neoplasia, which combined NRASQ61R oncogene expression in non-tumorous thyroid Nthy-ori 3-1 cells with a multi-dimensional proteomic technique DISER that consisted of profiling NRASQ61R-induced proteins using 2-dimensional difference gel electrophoresis (2D-DIGE) coupled with serological proteome analysis (SERPA) of the TAA repertoire of patients with thyroid encapsulated follicular-patterned/RAS-like phenotype (EFP/RLP) tumors. We identified several candidate cell-based (nicotinamide phosphoribosyltransferase NAMPT, glutamate dehydrogenase GLUD1, and glutathione S-transferase omega-1 GSTO1) and autoantibody (fumarate hydratase FH, calponin-3 CNN3, and pyruvate kinase PKM autoantibodies) biomarkers, including NRASQ61R-induced TAA phosphoglycerate kinase 1 PGK1. Meta-profiling of the reactivity of the identified autoantibodies across an independent SERPA series implicated the PKM autoantibody as a histological phenotype-independent biomarker of thyroid malignancy (11/38 (29%) patients with overtly malignant and uncertain malignant potential (UMP) tumors vs 0/22 (p = 0.0046) and 0/20 (p = 0.011) patients with non-invasive EFP/RLP tumors and healthy controls, respectively). PGK1 and CNN3 autoantibodies were identified as EFP/RLP-specific biomarkers, potentially suitable for further discriminating tumors with different malignant potential (PGK1: 7/22 (32%) patients with non-invasive EFP/RLP tumors vs 0/38 (p = 0.00044) and 0/20 (p = 0.0092) patients with other tumors and healthy controls, respectively; СNN3: 9/29 (31%) patients with malignant and borderline EFP/RLP tumors vs 0/31 (p = 0.00068) and 0/20 (p = 0.0067) patients with other tumors and healthy controls, respectively). The combined use of PKM, CNN3, and PGK1 autoantibodies allowed the reclassification of malignant/UMP tumor risk in 19/41 (46%) of EFP/RLP tumor patients. Taken together, we established an experimental pipeline DISER for the concurrent identification of cell-based and TAA biomarkers. The combination of DISER with in vitro oncogene expression allows further targeted identification of oncogene-induced TAAs. Using this integrated approach, we identified candidate autoantibody biomarkers that might be of value for differential diagnostic purposes in thyroid neoplasia.

Serum Immunoproteomics Combined With Pathological Reassessment of Surgical Specimens Identifies TCP-1ζ Autoantibody as a Potential Biomarker in Thyroid Neoplasia.

CONTEXT: Current methods of preoperative diagnostics frequently fail to discriminate between benign and malignant thyroid neoplasms. In encapsulated follicular-patterned tumors (EnFPT), this discrimination is challenging even using histopathological analysis. Autoantibody response against tumor-associated antigens is a well-documented phenomenon with prominent diagnostic potential; however, autoantigenicity of thyroid tumors remains poorly explored. OBJECTIVES: Objectives were exploration of tumor-associated antigen repertoire of thyroid tumors and identification of candidate autoantibody biomarkers capable of discrimination between benign and malignant thyroid neoplasms. DESIGN, SETTING, AND PATIENTS: Proteins isolated from FTC-133 cells were subjected to two-dimensional Western blotting using pooled serum samples of patients originally diagnosed with either papillary thyroid carcinoma (PTC) or EnFPT represented by apparently benign follicular thyroid adenomas, as well as healthy individuals. Immunoreactive proteins were identified using liquid chromatography-tandem mass-spectrometry. Pathological reassessment of EnFPT was performed applying nonconservative criteria for capsular invasion and significance of focal PTC nuclear changes (PTC-NCs). Recombinant T-complex protein 1 subunitζ (TCP-1ζ) was used to examine an expanded serum sample set of patients with various thyroid neoplasms (n = 89) for TCP-1ζ autoantibodies. All patients were included in tertiary referral centers. RESULTS: A protein demonstrating a distinct pattern of EnFPT-specific seroreactivity was identified as TCP-1ζ protein. A subsequent search for clinicopathological correlates of TCP-1ζ seroreactivity revealed nonclassical capsular invasion or focal PTC-NC in all TCP-1ζ antibody-positive cases. Further studies in an expanded sample set confirmed the specificity of TCP-1ζ autoantibodies to malignant EnFPT. CONCLUSIONS: We identified TCP-1ζ autoantibodies as a potential biomarker for presurgical discrimination between benign and malignant encapsulated follicular-patterned thyroid tumors. Our results suggest the use of nonconservative morphological criteria for diagnosis of malignant EnFPT in biomarker identification studies and provide a peculiar example of uncovering the diagnostic potential of a candidate biomarker using incorporation of pathological reassessment in the pipeline of immunoproteomic research.